Research Article International Ayurvedic Medical Journal ISSN:2320 5091

ANTIOXIDANT ACTIVITY OF “KRISHNA TILA”

1
Chougule Archana S., 2Thosar Chandrasekhar S.
1
MD (Ayu) , Shree Sai Baba Clinic, Degaon, 2MD (Scholar) , Shree Sai Baba Clinic,
Degaon
Email: archanachougule7@gmail.com

ABSTRACT
Antioxidant is a property of the drug that slows down the oxidation of hydrocarbons,
oils, fats etc & thus it helps to check the deterioration. Antioxidants work by significantly slow-
ing down the oxidation by the substance called ‘Free Radicals’. Krishna tila (Sesamum indicum,
Linn) is one of the plant origin drugs which had been mentioned for its rasayana properties in
classical literature of Ayurveda. So study was undertaken to evaluate the efficacy of Krishna Tila
in the form of oil , in the experimental study on healthy human blood serum regarding Antioxi-
dant activity.
Key words: Krishna tila , Antioxidant activity

INTRODUCTION:
Oxygen water and food are of fundamental im-
portance of life. Among these deprivation of
oxygen leads to sudden death. Oxygen is most
essential part of functions like metabolism, res-
piration, phagocytosis etc, but oxygen also plays
critical role in cellular injury by toxic activated
oxygen species which are called as “Free radi-
cals”. Free radicals are molecules or fractions of
molecule having unpaired electron in their outer
most orbit .Free radicals produced continuously
in cells during metabolism, respiration and de-
liberately during phagocytosis. These cytotoxic
free radicals play an important role in immune
system dysfunction which is responsible for
many disorders like arthritis, ulcerative colitis,
asthma, allergy, parasitic diseases, cancer and
cardiovascular disease.
Anti-oxidants are molecules that are
useful in preventing early oxidation of body
cells.. Antioxidants are the agents which
stimulates immunity of an individual either
in healthy or with immune deficiency.
Ayurveda is one among secular bio-
medical sciences. It is based on biophysical,
biochemical, physiological and bio-
pharmacodynamic principles. So it is said as
‘Rasa Rasayana chikitsa’. Rasayanas are the
groups herbal and herbo-mineral prepara-
tions explained in ayurvedic classics to pre-
vent the process of aging and to prevent the
diseases. These will help for the nourish-
ment of dhatus from rasa to shukra ultimate-
ly the ojas, which is responsible for physical
and psycho-intellectual performances. In
this way rasayana promotes long span of
youthful life full of vigor and free from dis-
eases and adverse effects of ageing. There-
fore it can be postulated that rasayana drugs
may possess Free radical scavenging proper-
ty.
Hence to provide a scientific data
and statistical validation a study on ‘Screen-
ing of free radical scavenging activity effect
of ‘Tila’ was under taken.
AIMS AND OBJECTIVES:
 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”
To evaluate antioxidant activity of
Krishna tila beeja (Sesamum indicum ,
Linn) through lipid per oxidation (
TBARS method).
REVIEW:
CONCEPT OF FREE RADICLE: 1,2
Definition: A molecule or molecular frag-
ment containing unpaired electron in va-
lence shell and capable of existing inde-
pendently is called ‘free radical. The mole-
cule of a compound is usually made up of
two parts which are known as radicals. For
example, the radicals present in sodium
chloride molecule are sodium and chloride.
These posses electric charge. So these are
highly reactive.
Types of free radicals: Free radicals may
be
 Oxygen derived ( Reactive Oxygen
Species, ROS )
 Nitrogen derived (Reactive Nitrogen
species, RNS )
 Sulphur derived ( Thinly / Thiele
free radicals ) and others.
Exogenous sources of ROS : Electromag-
netic radiation, cosmic rays, cigarette smok-
ing, car exhaust, UV light, Ozone, ionizing
radiation, certain drugs.
Endogenous sources of ROS : Mitochon-
drial electron transport chain, Respiratory
burst by phagocytosis, β oxidation of fat,
auto oxidation of amino acids, ischemia
reperfusion injury.
Free radical mediation of cell injury: One im-
portant mechanism of cell damage is injury in-
duced by free radicals, particularly by activated
oxygen species.
1. Lipid peroxidation of membranes
2. Mitochondrial damage
3. DNA damage
4. Protein damage
PHYSIOLOGICAL IMPORTANCE OF
FREE RADICALS: 3
Free radicals are produced continu-
ously in the cells as accidental by products
of metabolism, respiration and deliberately
during phagocytosis. Free radicals are natu-
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rally produced in the body through the nor-
mal metabolism of amino acids and fats. In
case of phagocytosis the bacteria to be
phagocytosed are first coated with antibody
(IgG) and compliment (C3), etc. This pro-
cess is called opsonisation. The phagocytic
cell then binds to this opsonised bacteria and
phagocyte is activated this leads to increased
movement and endocytosis. Release of en-
zymes and increased oxygen consumption
(Respiratory burst) forms toxic substance
like O2ˉ, H2O2, HOCl, NO which will kill the
bacteria. Subsequently endocytosed materi-
als are digested.
ANTI-OXIDANTS : 4
An antioxidant is a molecule capable
of slowing or preventing the oxidation of
other molecules. Oxidation is a chemical
reaction that transfers electrons from a sub-
stance to an oxidizing agent. Oxidation reac-
tions can produce free radicals, which start
chain reactions that damage cells. Antioxi-
dants terminate these chain reactions by re-
moving free radical intermediates, and in-
hibit other oxidation reactions by being oxi-
dized themselves. As a result, antioxidants
are often reducing agents.
Classification of Anti-oxidants:
1. Antioxidants are classified into two broad
divisions, depending on whether they are
soluble in water (hydrophilic) or in lipids
(hydrophobic).
2. a) Endogenous anti-oxidants: 5 These are
the physiological enzymes which are in-
volved in cellular defense against uncon-
trolled oxidative process.
b) Exogenous anti-oxidants: Those which
cannot be produced by human body, but
may protect pro-oxidant forces when admin-
istered. Such as Vitamin-E, Vitamin-C, β –
Carotene, lycopene, Selenium, Manganese,
copper, Zinc.
3. According to action: 6
a) Preventive anti-oxidants
b) Chain breaking anti-oxidants
c) Pharmacological anti-oxidants:
MEANING OF RASAYANA: 7
 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”
The word Rasayana is composed of
two words Ras + Ayan. The means by which
one gets the excellence of Rasa (The nour-
ishing fluid which is produced immediately
after digestion) is known as Rasayana.
Rasayana is a specialized type of
treatment influencing the fundamental as-
pect of body viz. Dhatus, Agni and Srotansi
and ojus etc. Rasayana Chikitsa boosts the
ojus and immune system. The adjective
Ojaswiis used to describe those people who
keep good health in all seasons and all stag-
es of life. It is like obtaining a high rank in a
physical or mental fitness. Ojus gives a
bright look, sharp memory, high perfor-
mance and every expected pleasure.
RASAYANA & ANTIOXIDANTS:8
Rasayana is one of the eight branch-
es of Ayurveda. The world Rasayana made
by worlds Rasa + Ayana. The world Rasa
has multiple meanings. It means shrangaara,
visha, veerya, guna, raga, drava etc. Ayana
means the pathway. So literally Rasayana
means the pathway to to get best quality of
dhatus. It helps to maintain the intactness of
body components also cures and prevents
the diseases.
ACTION OF RASAYANA:
Generally action of rasayana can be ex-
plained in three levels.
1. Poshak stara: Some of the rasayana act
as Rasa vardhaka. They mix with poshak
rasa and does the nourishment of dhatus. Ex-
Shatavari, Dugdha, Ghrita etc.
2. Agni stara: Some rasayana act on jatha-
ragni and other agni’s.They activate agni
and increase metabolism and help dhatu’s
for nourishment. Ex- Pippali rasayana.
3. Srotas stara: Some rasayana act on srotas
cleanses channels and help for the circula-
tion of rasa. Thus helps for the nourishment
of dhatus.Ex- Guggulu.
In modern view rasayana dravyas
work as anti-oxidants.. Achara rasayana act
as antioxidants, reduces the stress and thus
prevent the release of free radicles
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RAKTA DHATU IN AYURVEDA: 9
Rakta is originated from rasa dhatu. Rakta is
formed by raktagni through the prasadamsha of
rasa dhatu.
SERUM: 10
In blood, the serum is the component that is
neither a blood cell (serum does not contain white or
red blood cells) nor a clotting factor; it is the blood
plasma with the fibrinogens removed. Serum in-
cludes all proteins not used in blood clotting (coagu-
lation) and all the electrolytes, antibodies, antigens,
hormones, and any exogenous substances (e.g.,
drugs and microorganisms).The study of serum is
serology and may also include proteomics. Serum is
used in numerous diagnostic tests, as well as in
blood typing
VITAMIN C (standard drug) : 11,12 Vitamin C,
also known as ascorbic acid, is a water-soluble vita-
min which is essential for normal functioning of the
body.
CCL4 (Inducing drug ) 13
Synonyms: IUPAC Name -Tetra chloro-
methane
-1, 1, 1, 1-Tetrachloromethane
EXPERIMENTAL STUDY: 14
ANTIOXIDANT ACTIVITY:
Aim: To evaluate the antioxidant effect of
Krishna Tila (Sesamum indicum, Linn)
METHOD:
PARAMETERS TAKEN FOR ASSES-
MENT:
LIPID PEROXIDATION ( TBARS
METHOD) :
METHOD IN GENERAL:
 To take healthy human blood sample
& separation of serum
 Addition of drugs to serum & to ac-
cess the parameters
MATERIALS REQUIRED FOR LIPID
PEROXIDATION:
The lipid peroxidation of the test
sample is assessed by its absorption which
react with the Thiobarbituric acid & thus
produces the result. The protein in the serum
undergoes lipid peroxidation.
1. Drug : Krishna Tila Taila
 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”

2. Healthy human blood sample with the  20 % Trichloroacetic acid: 20 gms of

separation of serum TCA was dissolved in 100 ml of distilled
3. Equipments & glasswares water and kept in a bottle.
CHEMICALS & REAGENTS:  0.05 N sulphuric acid: 1.23 ml of conc.
 20% Trichloro acetic acid (TCA ) H2SO4 was diluted with 250 ml of dis-
 0.05N sulphuric acid tilled water & kept in bottle.
 2 molar sodium sulphate  2M sodium sulphate : 28.4 gms of unhy-
 Thibarbituric acid reagen drous sodium sulphate was dissolved in
 N- Butyl alcohol 90 ml of distilled water by heating &
METHODS: stirring. Then volume was made up to
LIPID PEROXIDATION (TBARS): 100 ml with distilled water.
Principle:  Thiobarbituric acid reagent: 0.67 gms
The proteins in serum are precipitat- (670 mg) of TBA was dissolved in 2M
ed by trichloroacetic acid (TCA) and the sodium sulphate solution by heating &
mixture is heated with thiobarbituric acid in the volume was made upto 100 ml, kept
2 M. sodium sulphate, in a boiling water in brown coloured bottle.
bath for 30 mins. The resulting chromogen 2) Preparation of Serum:
The blood from the healthy person is
Con- CCl4 Vit- C Test Test Test taken & is been kept in centrifugation
trol Treat- Treat- Dru Dru Dru chamber for the separation of the serum.
Group ed ed g g g This is used for the assessment.
1% 2% 5% 3) Method of preparation of Test Sample:
0.647 1.134 0.455 1.02 0.77 0.60 The fixed oil from the sesamum
5 5 8 seeds is been extracted through the soxhlet
0.583 1.108 0.467 1.04 1.06 0.82 apparatus & the solvent used is petroleum
4 4 1 ether. Hence the oil obtained is taken as the
0.756 1.211 0.666 0.87 0.58 1.00 test sample.
8 3 0 4) Method of Observation of absorption :
0.654 1.121 0.487 0.67 0.77 0.66  1 ml of T.B.A. reagent was added to
3 8 6 each dilutions (Test tube).
0.596 1.243 0.608 1.00 1.01 0.79  Kept in boiling water bath for 30 min for
6 9 4 coupling of lipid peroxide with TBA-
reagent.
is extracted with n-Butyl alcohol and ab-
 Pink color starts appearing in this stage.
sorbance of organic phase is determined at
 Then cooled in cold water
530 nm wavelength. The values are ex-
 Resulting chromogen extracted with 4
pressed in term of absorption of each sample
ml of n-Butyl alcohol by vigorous shak-
which is calculated by the Beer- Lamberts
ing.
Law.
 Then centrifuged at 3000 rpm for 10
Method of assessing Lipid peroxidation:
mins for the separation of organic phase
1. Preparation of Chemical Reagents
 Absorbance is Measured at 530 nm
2. Separation of serum from blood
wavelength
3. preparation of Test Sample
 The individual samples absorption are
4. observance of color change in different
calculated with Beer- Lamberts law for-
samples
mula.
5. Estimation of serum lipid peroxide
This formula is applied to calculate the mo-
1) Method of Preparation of Chemical
lar concentrations of each sample separately.
Reagents:
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 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”

5) Estimation of serum lipid per day 0.5ml of reaction mixture from the ho-
oxidation: mogenate which is kept for incubation is
Procedure: Aliquots of 4ml of ho- taken in a test tube and added with 4ml of
mogenate were taken in 2 different 25ml 10% Trichloroacetic acid( TCA). Contents
conical flasks. All the test tubes are added centrifuged at 4000 rpm for 10 min. 2ml of
with 6ml of potassium sulphate buffer ( pH, clear supernatant is taken in a graduated
7.4 ) , 8mlof 0.15ml potassium chloride. In tube. 2 ml of 0.67% Thiobarbituric acid
test group three different conical flasks add- (TBA) is added and heated in boiling water
ed three concentrations of drug like 1%, 2% bath for 15 min. Then tubes are cooled, pH
& 5% . In control group distilled water is is adjusted to 12-12.5.Colour which is de-
added, in standard group vit-c is addd, in veloped stabilized. Absorbance is measured
inducing group ccl4 is added & in test group at 540 nm in an UV spectrometer & calcu-
test sample is added. Totally 6 test tubes are lated by using Beer- Lamberts Law.
incubated at 37⁰c in incubator. Control group treated with: Distilled water
Lipid peroxidation is assessed on 1 st day( Induced group treated with : CCl4
after 45 min), 2 nd day and 4 th day.On each Standard drug treated with : Vit- C
Test group treated with : Krishna tila taila
OBSERVATIONS:
OBSERVATION – ANTIOXIDANT EFFCT OF Sesamum indicum:
OBSERVATION OF EXPERIMENTAL STUDY:
The molar concentrations of the samples are : Table no 10 LIPID
PROFILE OF THE SAMPLES:
1. CONTROL GROUP: Table no 11
Control group LPO in µM
concentrations
A 0.647
B 0.583
C 0.756
D 0.654
E 0.596
2. INDUCED GROUP : Table no 12

CCl4 treated LPO in µM

concentrations
A 1.134
B 1.108
C 1.211
D 1.121
E 1.243
3. STANDARD GROUP: Table no 1

Vit-C treated LPO in µM

concentrations
A 0.455
B 0.467
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 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”

C 0.666
D 0.487
E 0.608
4. 1% CONCENTRATION : Table no 14

1% concentration treated LPO in µM

concentrations
A 1.000
B 1.044
C 0.878
D 0.673
E 1.006

2% concentration treated LPO in µM

concentrations
A 0.775
B 1.064
C 0.583
D 0.788
E 1.019
5. 2% CONCENTRATION: Table no 15
6. 5% CONCENTRATION : Table no 16
5% concentration treated LPO in µM
concentrations
A 0.608
B 0.821
C 1.000
D 0.666
E 0.794

GRAPHICAL REPRESENTATION OF RESULTS:

1.5
Normal Control
CCl4 Treated
mM/mg Protein

1.0 Vitamin-C
**
** Test drug 1%
Test drug 2%
*** Test drug 5%
0.5

0.0
TBARS

DISCUSSION ON FREE RADICALS: Free radicals are molecules or frac-

tions of molecule having unpaired electron in

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 Chougule Archana S & Thosar Chandrasekhar S.Antioxidant Activity Of “Krishna Tila”
their outer most orbit .Free radicals produced
continuously in cells during metabolism, res-
piration and deliberately during phagocytosis.
These cytotoxic free radicals play an im-
portant role in immune system dysfunction
which is responsible for many disorders like
arthritis, ulcerative colitis, asthma, allergy,
parasitic diseases, cancer and cardiovascular
disease.
DISCUSSION ON ANTIOXIDANT PROP-
ERTY:
Anti-oxidants are molecules that are
useful in preventing early oxidation of body
cells. Oxidation of body cells can lead to
generation of free radicals that are solely
responsible for aging as well as degenerative
process happening in the body these anti-
oxidants are involved in inhibiting the pro-
duction of free radicals there by making the
person feel younger and healthier both men-
tally and physically. Antioxidants are the
agents which stimulates immunity of an in-
dividual either in healthy or with immune
deficiency. These produce effect either by
cellular or humoral or by both.
DISSCUSION ON ACTIVITY:
The result is been withdrawn on the
basis of the color change of the samples to
pink when TCA is added. This helped to es-
timate the overall lipid per oxidation in the
samples. And the same is been calculated
through absorption factor which is calculat-
ed by Beer- Lamberts law.
PROBABLE MODE OF ACTION:
The Rasayana (particularly those
with madhur vipaka that are advocated as
adapt gens in Ayurveda) primarily activate
immune cells, leading to secretion of cyto-
kines, which in turn act on multiple target
organs to produce the myriad effects as-
cribed to these treatments.
It has been found that the nervous,
endocrine and immune systems are all inter-
related. Immune products like various cyto-
kines have been found to stimulate the hypo-
thalamus-pituitary-adrenal axis and cortico-
trophin release factor (CRF), which ulti-
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mately enhances the production of adrenal
corticotrophic hormone (ACTH) resulting
into increased secretion of glucocorticoids
which have an overall suppressive effect on
the immune system. Stress also acts on the
same axis and brings about changes in the
immune status of the body. These Rasayana
drugs probably reduce stress levels by af-
fecting antioxidant levels. So these Ra-
sayana drugs act as potent antioxidants.
CONCLUSIONS:
1. Madhura, tikta, kashaya –rasa, madhura-
vipaka , guru, snigdha- gunas, these
qualities in Krishna tila acts as rasayana.
2. Presence of flavonides & tannins in
Krishna tila beeja dominates antioxidant
property.
3. Study has shown significant antioxidant
activity on molar concentrations of 2%
& 5%.
4. Easily available Krishna tila can be
used safely as substitute over modern an-
tioxidant
drugs.
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Source of support: Nil

Conflict of interest: None Declared

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